The long-term objective of this work is to understand the mechanism of synaptic transmission in sensory receptor cells in the ear, eye and other sensory systems that use graded transmission at ribbon-class synapses. This specialized form of chemical signaling appears to be an adaptation to allow the transmission of information about small changes in sensory input that would be lost during conventional synaptic transmission that uses action potentials. The proposed experiments will investigate the physiology, anatomy and biochemistry of these synapses. The work on sensory receptors (hair cells) in the ear will use frogs and zebrafish as model species. The zebrafish work will also study photoreceptors and bipolar cells in the retina, and hair cells in the lateral line organs. All of these calls have ribbon-class synapses. These species were chosen for study because of the cellular and molecular tools that are available to answer fundamental questions in synaptic physiology. In the case of frogs, there is also a wealth of information already available upon which to build. During the past 10 years, several laboratories have developed techniques that allow detailed electrophysiological analysis of synaptic transmission using tight-seal voltage clamp to measure small changes in membrane capacitance to observe synaptic transmission on a millisecond time scale. This physiological method will be used in conjunction with electron tomography and recently developed membrane tracer dyes to study the ultra structure of the synaptic vesicle cycle ribbon synapses. The goal is to test several key hypotheses concerning the function of the synaptic "ribbon", the prominent anatomical feature for which these synapses are named. The project will also focus on two major proteins (calretinin and parvalbumin 3) that are believed to serve central roles in synaptic transmission in these cells by capturing and transporting calcium ions away from the synapses. The project will investigate the important biochemical properties that determine how fast these proteins bind calcium, how much calcium they can sequester, and how fast they can diffuse within the cell. These properties are central to understanding synaptic transmission in hair cells, and have a wider relevance to the proposed function of these and related calcium-binding proteins in protection from calcium overload during strokes and other brain injuries. The genetic and molecular tools developed by zebrafish researchers during the past decade will allow a direct test the function of a protein (Ribeye) that has recently been identified as a major component of the ribbon.